The goal of the research to be carried out under this fellowship is to understand the mechanisms that lead to endothelial dysfunction in hypercholesterolemia. Nitric Oxide(NO) is the most potent vasodilator in the cardiovascular system, in the disease state, the bioavailability of NO is decreased and this is thought to play a critical role in the initiation of atherosclerosis and increases the risk associated with other cardiovascular diseases. We hypothesize that this decrease in NO is due to an increase in cellular methylarginine levels. Methylarginines, ADMA and NNMA, are competitive inhibitors of NO production and compete with L-Arg for NOS binding. Normally methylarginines are metabolized by the enzyme DDAH, however, initial cellular studies have shown that DDAH activity is lost when endothelial cells are exposed to either oxidized LDL (OxLDL) or the lipid hydroperoxide, 4-hydroxynonenal (HNE). Therefore, the aim of the current proposal is to test the hypothesis that elevations in oxidatively modified lipids such as oxLDL and HNE, inhibit eNOS activity and cause endothelial dysfunction through increased intracellular accumulation of the endogenous methylarginines ADMA and L-NMMA. In aim 1 of this study we will examine the effects of oxLDL and other lipid hydroperoxides on endothelial DDAH expression and NO production in endothelial cells. In aim 2 we will look at the effects of DDAH modulation on the initiation and progression of atherosclerosis in hypercholesterolemic/pro-atherogenic LDL receptor knockout mice. For each of these aims, EPR measurements of NO and .02- will be combined with cellular, molecular and physiological approaches to characterize the effects of DDAH modulation on NOS function and NO production using in vitro and in vivo models. Results from these studies will provide fundamental mechanistic information regarding the effects of modified lipids in NOS regulation and This work will help to establish the role of DDAH in the progression of atherosclerosis and perhaps lead to DDAH being used as a therapeutic target to help alleviate the risk associated with this disease and other cardiovascular disorders.